Most organisms have an absolute requirement for iron. The scarcity of free iron in vertebrate hosts may limit infection and invasion by pathogenic microorganisms. Thus, competition for this element plays a pivotal role in host-bacterial interactions. Reduced levels of available iron in the host not only limit bacterial growth but also act as a regulatory signal to induce synthesis of iron acquisition systems, certain toxins, and other proteins associated with virulence. Thus iron is one of a number of environmental stimuli to which pathogens respond. The broad objectives of this project are to (A) study the structure and regulation of bacterial iron transport genes, (B) determine the mechanism of iron acquisition in vivo, and (C) study the genes and gene products which are differentially expressed in the host and determine the signals which, in addition to iron, regulate their expression. The model system for these studies is the enteric pathogen Shigella flexneri. This pathogen causes disease by invading and multiplying within intestinal epithelial cells, and thus is a model for studying invasive, intracellular pathogens. S. flexneri is readily amenable to genetic and biochemical analysis, and tissue culture can be used to study the interaction of these bacteria with host cells. The specific goals of this proposal are to 1) determine the mechanism by which Shigella utilize heme as a sole source of iron, 2) determine the role of the heme-iron transport system in virulence, and 3) measure the expression of iron transport and other virulence-associated genes within host cells. Genetic and biochemical approaches will be used to study heme transport. The genes encoding the system will be cloned, and mutants will be isolated to study gene function and regulation. Those mutants defective in binding or transport of heme will also be tested for invasion and intracellular multiplication in tissue culture systems. In addition to in vitro analysis, techniques have been developed to measure protein expression by S. flexneri invading or multiplying within host cells. This provides a tool for determining expression of virulence factors in vivo and, in the long term, will be a useful technique in design and analysis of potential vaccine strains.